The mitogen activated protein kinase (MAPK) signaling pathways are involved in cellular events such as growth, differentiation and stress responses (J. Biol. Chem. (1993) 268, 14553-14556). Four parallel pathways have been identified to date: ERK1/ERK2, JNK, p38 and ERK5. These pathways are linear kinase cascades in that MAPKKK phosphorylates and activates MAPKK that phosphorylates and activates MAPK. To date, there are 7 MAPKK homologs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK6, and MKK7) and 4 MAPK families (ERK1/2, JNK, p38, and ERK5). The MAPKK family members are unique in that they are dual-specific kinases, phosphorylating MAPKs on threonine and tyrosine. Activation of these pathways regulates the activity of a number of substrates through phosphorylation. These substrates include transcription factors such as TCF, c-myc, ATF2 and the AP-1 components, fos and Jun; the cell surface components EGF-R; cytosolic components including PHAS-I, p90.sup.rsk, cPLA.sub.2 and c-Raf-1; and the cytoskeleton components such as tau and MAP2.
The prototypical mitogen activated protein kinase cascade is reflected by the ERK pathway (Biochem J. (1995) 309, 361-375). The ERK pathway is activated primarily in response to ligation of receptor tyrosine kinases (RTKs) (FEBS Lett. (1993) 334, 189-192). Signal propagation from the RTKs occurs down the Ras pathway through sequential phosphorylation of Raf, MEK and ERK. This pathway has not been typically viewed of as an important contributor to the inflammatory response, but rather involved in growth and differentiation processes. This view stems from the profile of typical activators of this pathway, which include growth factors (PDGF, NGF, EGF), mitogens (phorbol esters), and polypeptide hormones (insulin, IGF-1). Evidence for ERK pathway involvement in inflammatory and immune responses has, however, gained some support in recent years (Proc. Natl. Acad. Sci. USA. (1995) 92, 1614-1618; J. Immunol. (1995) 155, 1525-1533; J. Biol. Chem. (1995) 270, 27391-27394; and Eur. J. Biochem. (1995) 228, 1-15). Cytokines such as TNFa and IL-1b, the bacterial cell wall mitogen, LPS, and chemotactic factors such as fMLP, C5a, and IL-8 all activate the ERK pathway. In addition, the ERK pathway is activated as a result of T cell receptor ligation with antigen or agents such as PMA/ionomycin or anti-CD3 antibody, which mimic TCR ligation in T cells (Proc. Natl. Acad. Sci. USA (1995) 92, 7686-7689). These findings indicate that inhibitors of the ERK pathway should function as anti-inflammatory and immune suppressive agents.
Small molecule inhibitors of the Raf/MEK/ERK pathway have been identified. A series of benzoquinones has been disclosed by Parke-Davis, which is exemplified by PD 098059 that inhibits MEK activity (J. Biol. Chem. (1995) 46, 27498-27494). Recently, we identified a MEK inhibitor, U0126 (J. Biol. Chem. (1998) 29, 18623-18632). Comparative kinetic analysis showed that U0126 and PD 098059 were non-competitive inhibitors of activated MEK (J. Biol. Chem. (1998) 29, 18623-18632). These MEK inhibitors have been used to investigate the role of the ERK activation cascade in a wide variety of systems including inflammation, immune suppression and cancer. For example, PD 098059 blocks thymidine incorporation into DNA in PDGF-stimulated Swiss 3T3 cells (J. Biol. Chem. (1995) 46, 27498-27494). PD 098059 also prevents PDGF-BB-dependent SMC (Smooth Muscle Cell) chemotaxis at concentrations which inhibit ERK activation (Hypertension (1997) 29, 334-339). Similarly, U0126 prevents PDGF-dependent growth of serum starved SMC. We have also shown that U0126 blocks keratinocyte proliferation in response to a pituitary growth factor extract, which consists primarily of FGF. These data coupled with those obtained with PD 098059 above indicate that MEK activity is essential for growth factor-stimulated proliferation.
The role of the MEK/ERK pathway in inflammation and immune suppression has been examined in a number of systems, including models of T cell activation. The T cell antigen receptor (TCR) is a non-RTK receptor whose intracellular signaling pathways have been elucidated (Proc. Natl. Acad. Sci. USA (1995) 92, 7686-7689). DeSilva et al. have generated a great deal of information with U0126 in T cell systems (J. Immunol. (1998) 160, 4175-4181). Their data showed that U0126 prevents ERK activation in T cells in response to PMA/ionomycin, Con A stimulation, and antigen in the presence of costimulation. In addition, T cell activation and proliferation in response TCR engagement is blocked by U0126 as is IL-2 synthesis. These results indicate that MEK inhibition does not result in a general antiproliferative effect in this IL-2-driven system, but selectively blocks components of the signaling cascades initiated by T cell receptor engagement.
PD 098059 has also been shown to inhibit T cell proliferation in response to anti-CD3 antibody, which is reversed by IL-2 (J. Immunol. (1998) 160, 2579-2589). PD 098059 also blocked IL-2 production by T cells stimulated with anti-CD3 antibody in combination with either anti-CD28 or PMA. In addition, the MEK inhibitor blocked TNFa, IL-3 GM-CSF, IFN-g, IL-6 and IL-10 production. In contrast, PD 098059 enhanced production of IL-4, IL-5 and IL-13 in similarly stimulated T cell cultures. These differential T cells effects with MEK inhibition suggest that therapeutic manipulations may be possible.
Neutrophils show ERK activation in response to the agonists N-formyl peptide (fMLP), IL-8, C5a and LTB.sub.4, which is blocked by PD 098059 (Biochem. Biophy. Res. Commun. (1997) 232, 474-477). Additionally, PD 098059 blocks neutrophil chemotaxis in response to all agents, but does not alter superoxide anion production. However, fMLP-stimulated superoxide generation was inhibited by PD098059 in HL-60 cells (J. Immunol. (1997) 159, 5070-5078), suggesting that this effect may be cell-type specific. U0126 blocks ERK activation in fMLP- and LTB.sub.4 -stimulated neutrophils, but does not impair NADPH-oxidase activity or bacterial cell killing. U0126 at 10 mM blunts up regulation of b2 integrin on the cell surface by 50% and blocks chemotaxis through a fibrin gel &gt;80% in response to IL-8 and LTB.sub.4. Thus, neutrophil mobility is affected by MEK inhibition although the acute functional responses of the cell remain intact.
Eicosanoids are key mediators of the inflammatory response. The proximal event leading to prostaglandin and leukotriene biosynthesis is arachidonic acid release from membrane stores, which is mediated largely through the action of cytosolic phospholipase A.sub.2 (cPLA.sub.2). Activation of cPLA.sub.2 requires Ca.sup.2+ along with phosphorylation on a consensus MAP kinase site, Ser.sup.505, which increases catalytic efficiency of the enzyme (J. Biol. Chem. (1997) 272, 16709-16712). In neutrophils, mast cells, or endothelial cells, PD 098059 blocks arachidonic acid release in response to opsonized zymosan, aggregation of the high affinity IgG receptor, or thrombin, respectively. Such data support a role for ERK as the mediator of cPLA.sub.2 activation through phosphorylation (FEBS Lett. (1996) 388, 180-184; Biochem J. (1997) 326, 867-876; and J. Biol. Chem. (1997) 272, 13397-13402). Similarly, U0126 is able to block arachidonic acid release along with prostaglandin and leukotriene synthesis in keratinocytes stimulated with a variety of agents. Thus, the effector target, cPLA2, is sensitive to MEK inhibition in a variety of cell types.
MEK inhibitors also seem to affect eicosanoid production through means other than inhibition of arachidonic acid release. PD 098059 partially blocked LPS-induced Cox-2 expression in RAW 264.7 cells, indicating ERK activation alone may not be sufficient to induce expression of this key enzyme mediating inflammatory prostanoid production (Biochem J. (1998) 330, 1107-1114). Similarly, U0126 inhibits Cox-2 induction in TPA-stimulated fibroblasts, although it does not impede serum induction of the Cox-2 transcript. PD 098059 also inhibits Cox-2 induction in lysophosphatidic acid (LPA)-stimulated rat mesangial cells, which further supports a role for ERK activation in production of prostaglandins (Biochem J. (1998) 330, 1107-1114). Finally, 5-lipoxygenase translocation from the cytosol to the nuclear membrane along with its activation as measured by 5-HETE production can be inhibited by PD 098059 in HL-60 cells (Arch. Biochem. Biophys. (1996) 331, 141-144).
Inflammatory cytokines such as TNFa and IL-1b are critical components of the inflammatory response. Cytokine production in response to cell activation by various stimuli as well as their activation of downstream signaling cascades represent novel targets for therapeutics. Although the primary effect of IL-1b and TNF-a is to up-regulate the stress pathways (Nature (1994) 372, 729-746), published reports (Proc. Natl. Acad. Sci. USA (1995) 92, 1614-1618; J. Immunol. (1995) 155, 1525-1533; J. Biol. Chem. (1995) 270, 27391-27394. Eur. J. Biochem. (1995) 228, 1-15.). Cytokines such as TNFa and IL-1b, the bacterial cell wall mitogen, LPS, and chemotactic factors such as fMLP, C5a, and IL-8 all activate the ERK pathway. In addition, the ERK pathway is activated as a result of T cell receptor ligation with antigen or agents such as PMA/ionomycin or anti-CD3 antibody, which mimic TCR ligation in T cells (Proc. Natl. Acad. Sci. USA (1995) 92, 7686-7689) and clearly show that the ERK pathway is also affected. U0126 can block MMP induction by IL-1b and TNF-a in fibroblasts (J. Biol. Chem. (1998) 29, 18623-18632), demonstrating that ERK activation is necessary for this proinflammatory function. Similarly, lipopolysaccharide (LPS) treatment of monocytes results in cytokine production that has been shown to be MAP kinase-dependent being blocked by PD 098059 (J. Immunol. (1998) 160, 920-928). Indeed, we have observed similar results in freshly isolated human monocytes and THP-1 cells where LPS-induced cytokine production is inhibitable by U0126 (J. Immunol. (1998) 161:5681-5686).
The proximal involvement of RAS in the activation of the ERK pathway suggests that MEK inhibition might show efficacy in models where oncogenic RAS is a determinant in the cancer phenotype. Indeed, PD 098059 (J. Biol. Chem. (1995) 46, 27498-27494) as well as U0126 are able to impede the growth of RAS-transformed cells in soft agar even though these compounds show minimal effects on cell growth under normal culture conditions. We have further examined the effects of U0126 on the growth of human tumor cell lines in soft agar. We have shown that U0126 can prevent cell growth in some cells, but not all, suggesting that a MEK inhibitor may be effective in only certain kinds of cancer. In addition, PD 098059 has been shown to reduce urokinase secretion controlled by growth factors such as EGF, TGFa and FGF in an autocrine fashion in the squamous cell carcinoma cell lines UM-SCC-1 and MDA-TV-138 (Cancer Res. (1996) 56, 5369-5374). In vitro invasiveness of UM-SCC-1 cells through an extracellular matrix-coated porous filter was blocked by PD 098059 although cellular proliferation rate was not affected. These results indicate that control of the tumor invasive phenotype by MEK inhibition may also be a possibility. The observed effects with PD 098059 and U0126 suggest that MEK inhibition may have potential for efficacy in a number of disease states. Our own data argue strongly for the use of MEK inhibitors in T cell mediated diseases where immune suppression would be of value. Prevention of organ transplant rejection, graft versus host disease, lupus erythematosus, multiple sclerosis, and rheumatoid arthritis are potential disease targets. Effects in acute and chronic inflammatory conditions are supported by the results in neutrophils and macrophage systems where MEK inhibition blocks cell migration and liberation of proinflammatory cytokines. A use in conditions where neutrophil influx drives tissue destruction such as reperfusion injury in myocardial infarction and stroke as well as inflammatory arthritis may be warranted. Blunting of SMC migration and inhibition of DNA replication would suggest atherosclerosis along with restenosis following angioplasty as disease indications for MEK inhibitors. Skin disease such as psoriasis provides another potential area where MEK inhibitors may prove useful since MEK inhibition prevents skin edema in mice in response to TPA. MEK inhibition also blocks keratinocyte responses to growth factor cocktails, which are known mediators in the psoriatic process. Finally, the use of a MEK inhibitor in cancer can not be overlooked. Ionizing radiation initiates a process of apoptosis or cell death that is useful in the treatment solid tumors. This process involves a balance between pro-apoptotic and anti-apoptotic signal (Science 239, 645647), which include activation of MAP kinase cascades. Activation of the SAPK pathway delivers a pro-apoptotic signal (Radiotherapy and Oncology (1998) 47, 225-232.), whereas activation of the MAPK pathway is anti-apoptotic (Nature (1996) 328, 813-816.). Interference with the anti-apoptotic MAPK pathway by dominant negative MEK2 or through direct inhibition of MEK with synthetic inhibitors sensitizes cells to radiation-induced cell death (J. Biol. Chem. (1999) 274, 2732-2742; and Oncogene (1998) 16, 2787-2796). Thus, a MEK would be useful as a radiosensitizer in the treatment of solid tumors.
U.S. Pat. No. 5,099,021 describes a process for the preparation of unsymmetrically disubstituted ureas, but does not include an adamantyl moiety.